Overexpressed GM1 Suppresses Nerve Growth Factor (NGF) Signals by Modulating the Intracellular Localization of NGF Receptors and Membrane Fluidity in PC12 Cells

• Published in: The Journal of biological chemistry Vol. 279; no. 32; pp. 33368 - 33378
• Main Authors: Nishio, Masashi, Fukumoto, Satoshi, Furukawa, Keiko, Ichimura, Akiko, Miyazaki, Hiroshi, Kusunoki, Susumu, Urano, Takeshi, Furukawa, Koichi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06-08-2004; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Dimerization; Enzyme Activation - drug effects; Fluorescent Antibody Technique; G(M1) Ganglioside - genetics; G(M1) Ganglioside - physiology; Galactosyltransferases - genetics; Ganglioside Galactosyltransferase; Gangliosides - genetics; Gene Expression; Glycosphingolipids - genetics; Humans; Membrane Fluidity - physiology; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases - metabolism; Nerve Growth Factor - antagonists & inhibitors; Nerve Growth Factor - metabolism; Nerve Growth Factor - pharmacology; Neurites - drug effects; Neurites - physiology; PC12 Cells; Phosphorylation; Rats; Receptor, Nerve Growth Factor - analysis; Receptor, Nerve Growth Factor - physiology; Receptor, trkA - chemistry; Receptor, trkA - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M403816200

Ganglioside GM1 has been considered to have a neurotrophic factor-like activity. To analyze the effects of endogenously generated GM1, the rat pheochromocytoma cell line PC12 was transfected with the GM1/GD1b/GA1 synthase gene and showed increased expression levels of GM1. To our surprise, GM1+-transfectant cells (GM1+ cells) showed no neurite formation after stimulation with nerve growth factor (NGF). Autophosphorylation of NGF receptor TrkA and activation of ERK1/2 after NGF treatment were scarcely detected in GM1+ cells. Binding of 125I-NGF to PC12 cells was almost equivalent between GM1+ cells and controls. However, dimer formation of TrkA upon NGF treatment was markedly suppressed in GM1+ cells in both cross-linking analysis with Bis(sulfosuccinimidyl)suberate 3 and 125I-NGF binding assay. The sucrose density gradient fractionation of the cell lysate revealed that TrkA primarily located in the lipid raft fraction moved to the non-raft fraction in GM1+ cells. p75NTR and Ras also moved from the raft to non-raft fraction in GM1+ cells, whereas flotillin and GM1 persistently resided in the lipid raft. TrkA kinase activity was differentially regulated when GM1 was added to the kinase assay system in vitro, suggesting suppressive/enhancing effects of GM1 on NGF signals based on the concentration. Measurement of fluorescence recovery after photobleaching revealed that the membrane fluidity was reduced in GM1+ cells. These results suggested that overexpressed GM1 suppresses the differentiation signals mediated by NGF/TrkA by modulating the properties of the lipid raft and the intracellular localization of NGF receptors and relevant signaling molecules.